Many chemicals that are currently derived from petrochemical materials could be produced from naturally occurring carbohydrates. In particular, succinic acid, a four-carbon dicarboxylic acid, has the potential to become a high volume commodity chemical that could be used as starting material for commercial processes that produce many important intermediate and specialty chemicals for the consumer product industries and that currently rely on starting materials derived from non-renewable petrochemical materials. For example, as a commodity chemical, succinic acid could replace petrochemical starting materials used in the production of 1,4-butanediol (BDO) and tetrahydrofuran (THF) compounds, which are useful as solvents and starting materials for many industries. For example, BDO and THF compounds are useful for producing resins for automotive bodies, thermoplastics for use in household appliances, and elastic polymers such as Lycra™ in the textile industry. In addition, BDO and THF compounds also have many specialty uses in the agrochemical and pharmaceutical industries. Notably, worldwide consumption of BDO is expected to increase at an annual rate as high as 4%.
The petrochemicals currently used to produce BDO and THF include acetylene, formaldehyde, butane, butadiene, and propylene oxide. All of these have various hazardous properties, such as extreme flammability, chemical instability and toxicity. Further, as these materials are derived from petroleum, they deplete a non-renewable resource, and upon disposal or destruction, ultimately release carbon (as carbon dioxide) into the atmosphere. Thus, developing succinic acid as a replacement for petrochemically derived materials would reduce handling and storage of hazardous materials, enhance industrial and community safety, reduce pollution and environmental costs, and reduce dependence on oil.
Production of succinic acid and other organic compounds by fermentation of sugars is economically feasible. A number of microorganisms have been used to produce succinic acid using corn sugars as a carbon source. As such, developing succinic acid as replacement for petrochemical starting materials would expand markets for corn, and other agricultural products and/or biomass that can provide fermentable sugars.
Formally, the biochemical pathway for succinic acid production adds a carbon dioxide molecule to the three carbon compound phosphoenolpyruvate (PEP), to produce the four carbon compound oxaloacetate (OAA). The next steps in the pathway to succinic acid are part of the reverse tricarboxylic acid cycle (TCA cycle) and include two obligate reduction steps. In the biochemical process leading from OAA to succinate, OAA must first be reduced to produce L-malate. L-malate is then dehydrated to produce fumarate and water. Fumarate is then reduced to give the succinic acid. In the chemical arts, “reduction” refers to the addition of molecular hydrogen to a compound.
Generally, free molecular hydrogen is not found in intracellular biological systems. Rather, reduction is performed through the use of coenzymes that function as biochemical equivalents of hydrogen (i.e., as carriers of molecular hydrogen) and are termed “reducing equivalents.” Reducing equivalents include the coenzymes nicotinamide adenine dinucleotide hydrogen (“NADH”), nicotinamide adenine dinucleotide phosphate hydrogen (“NADPH”), flavine adenine dinucleotide hydrogen (“FADH2”), and flavin mononucleotide hydrogen (“FMNH”). Generally, NADH and NADPH may be interconverted in a range of microorganisms by the enzyme pyridine dinucleotide transhydrogenase.
The reducing equivalents required to transform OAA to succinate are provided by NAD(P)H2, FADH2, or other co-factors. It is essential that a sufficient quantity of reducing equivalents is available for the transformation of OAA to succinate. If sufficient reducing equivalents are not available, the biochemical pathway will not function efficiently, and only a portion of the OAA will be transformed into the desired succinate.
Reducing equivalents may be produced in a number of biological processes that are commonly found in cellular metabolism. For example, reducing equivalents may be generated in the pentose phosphate cycle (PPC). In the PPC, glucose-6-phosphate is converted to D-6-phospho-glucono-δ-lactone by the enzyme glucose-6-phosphate dehydrogenase, which is also known as Zwischenferment enzyme or Zwf. As part of this conversion, NADP is converted to NADPH as an acceptor of reducing equivalents.
Few microorganisms have been described which produce sufficient concentrations of succinic acid for commercial production. One such microorganism is Actinobacillus succinogenes, a facultative anaerobe that was isolated from the bovine rumen. This organism produces high concentration of succinic acid and tolerates high sugar concentration. Actinobacillus succinogenes is one of the best known producers of succinic acid, but the fermentative yields of this strain may be limited by the lack of reducing equivalents. As such, improvements are desirable to increase the yield of succinic acid produced by fermentation, including the use of improved strains of microorganisms for producing succinic acid.